The Autographa californica M nucleopolyhedrovirus (AcMNPV) encodes a gene (open reading frame 32) with homology to vertebrate and invertebrate fibroblast growth factors (fgfs), key regulators of developmental processes affecting the growth, differentiation, and migration of many cell types. We studied the temporal regulation of the AcMNPV fgf, vfgf, by Northern (RNA) blot hybridization; vfgf was transcribed as a 0.6-kb mRNA at early times but as part of a 1.4-kb bicistronic mRNA at late times. The product of vfgf, vFGF, exhibited a number of characteristics that have also been demonstrated for other FGF homologs. vFGF had strong affinity to heparin, a property important for FGF signaling via an FGF receptor. vFGF was secreted into the extracellular fluid when expressed in insect cells, suggesting that it acts as an extracellular ligand. Finally, vFGF was able to stimulate migration of several different types of insect cells. We discuss how this activity may be important for its function during virus infection.

The Autographa californica M nucleopolyhedrovirus (AcMNPV) viral fibroblast growth factor (vFGF) has functional parallels to cellular FGFs. Deletion of the AcMNPV vfgf has no obvious phenotype in cell culture but delays the time of insect death. Here, we determined vFGF production during virus infection. vFGF was detected at 24 hours post infection and through the reminder of the infection cycle. Since vFGF is thought to be a secreted membrane-binding protein and virions acquire an envelope derived from the cell membrane, we examined virions for the presence of vFGF using microscopy, flow cytometry, and affinity chromatography. We found that vFGF associated with virions. Furthermore, budded virus carrying vFGF had more affinity to heparin than vFGF-deficient budded virus, consistent with the affinity of FGFs for heparan sulfate proteoglycans. Although the function of virion-associated vFGF is not clear, we found that virion-associated vFGF stimulated cell motility and affected virus attachment.

BackgroundHematophagous Culicoides spp. biting midges are of great agricultural importance as livestock, equine, and wildlife pests and as vectors of the orbiviruses bluetongue, epizootic hemorrhagic disease and African horse sickness. To obtain a blood meal, midges deposit saliva containing allergens, proteases, and anti-hemostatic factors, into the dermis to facilitate feeding. Infected midges deposit virus along with the myriad of salivary proteins during feeding. The extreme efficiency with which midges are able to transmit orbiviruses is not clearly understood, as much is still unknown about the physiological trauma of the bite and immune responses to saliva deposited during feeding. Of particular interest are the first few hours and days after the bite; a critical time period for any midge-transmitted virus to quickly establish a localized infection and disseminate, while avoiding the hosts’ immune responses.ResultsA mouse-midge feeding model using colonized Culicoides sonorensis midges was used to characterize innate mammalian immune responses to blood-feeding. Histological analysis of skin, and cellular and cytokine profiles of draining lymph nodes show Culicoides midge feeding elicited a potent pro-inflammatory Th-mediated cellular response with significant mast cell activation, subcutaneous hematomas, hypodermal edema and dermal capillary vasodilation, and rapid infiltration of leukocytes to the bite sites. Mast cell degranulation, triggered by bite trauma and specifically by midge saliva, was key to physiological and immunological responses and the ability of midges to feed to repletion.ConclusionsMidge feeding causes physiological and immunological responses that would be highly favorable for rapid infection and systemic dissemination orbiviruses if delivered during blood-feeding. Recruitment of leukocytic cells to bitten skin brings susceptible cell populations in proximity of deposited virus within hours of feeding. Infected cells would drain to lymph nodes, which become hyperplastic in response to saliva, and result in robust viral replication in expanding cell populations and dissemination via the lymph system. Additionally, saliva-induced vasodilation and direct breaches in dermal capillaries by biting mouthparts exposes susceptible vascular endothelial cells, thereby providing immediate sites of virus replication and a dissemination route via the circulatory system. This research provides insights into the efficiency of Culicoides midges as orbivirus vectors.

Autographa californica M nucleopolyhedrovirus (AcMNPV) open reading frame 109 (ac109) is conserved in all known baculovirus genomes, suggesting a crucial role in virus replication. Although viruses lacking ac109 have been previously characterized, the phenotypes differ from production of non-infectious virions to lack of virion production. To re-examine ac109 function, we constructed a recombinant AcMNPV bacmid, AcBAC109KO, with a deletion in ac109. We did not detect infectious budded virus after transfection of AcBAC109KO DNA into cells. In the nucleus, nucleocapsids had envelopment defects and polyhedra lacked virions. DNA synthesis and gene expression between AcBAC109KO and a control virus were similar. However, lower levels of non-infectious budded virus were detected from AcBAC109KO DNA-transfected cells compared to the parental virus using Q-PCR to detect viral DNA or by immunoblotting to detect a budded virus protein. Therefore, deletion of ac109 affects envelopment of nucleocapsids in the nucleus and the production of infectious budded virus.